CREATION OF DIFFERENTIALLY METHYLATED BACKGROUND GENE SET FOR GO ENRICHMENT

<br>SUMMARYThis script prepares a background gene set for GO enrichment analysis based on differentially methylated CpGs in <i>Nasonia vitripennis</i> from an ageing (diapause) methylation dataset. It filters CpGs, maps them to genomic features, and annotates them with gene identifiers (gene_id) using exon/intron reference data. GO annotations are then assigned to generate a background for downstream enrichment testing.<br><br>KEY STEPS1. Load Erin's methylation data (erindata_v2.txt)2. Filter for CpGs in the ageing-related DML list (timepoint DMLs)3. Extract chromosome and position from CpG row identifiers4. Join CpG positions with exon/intron gene annotations (Nvit genome)5. Retain only those CpGs overlapping gene features6. Assign GO terms to mapped gene_ids using Nasonia GO file7. Output the resulting gene-GO annotation file as a background set<br>INPUT FILES<br>- erindata_v2.txt: Erin's filtered methylation matrix- un.dmls.timepoint.txt: Differentially methylated CpG list (timepoint only)- GCF_009193385.2_Nvit_psr_1.1_genomic_numbered_exons.txt: Nasonia exon/intron annotation- Nasonia_PSR1.1_Gene_GO.txt: GO annotations for Nasonia genes<br>OUTPUT FILES<br>- diff_methylatedcpgs_with_LOC.csv: CpGs mapped to annotated gene features- diff_backgroundGOannotations.csv: Final gene-GO background used for enrichment<br>SOFTWARE REQUIREMENTS<br>- R packages: data.table, dplyr, tidyr, readr, sqldf, GOstats, GSEABase, treemap- Input genome annotation must contain numbered exon/intron coordinates and gene_id mappings<br>NOTES- GO evidence code is set to "IEA" for all terms- CpGs not overlapping gene features are excluded from the background- This background set is later used in GO enrichment scripts (see Table S4 analysis)<br><br><br>CONTACT<br>Eamonn Mallon [e-mail]<br>

摘要
本脚本基于衰老（滞育）甲基化数据集中的丽蝇蛹集金小蜂（Nasonia vitripennis）差异甲基化CpG位点（differentially methylated CpGs），为GO富集分析（GO enrichment analysis）构建背景基因集。流程包括过滤CpG位点、将其映射至基因组特征，并利用外显子/内含子参考数据为其注释基因标识符（gene_id）。随后分配GO注释，以生成用于下游富集测试的背景集。

关键步骤
1. 加载Erin的甲基化矩阵数据（erindata_v2.txt）
2. 从衰老相关差异甲基化位点（differentially methylated loci, DML）列表中筛选CpG位点（仅时间点相关DML）
3. 从CpG行标识符中提取染色体编号与位点位置
4. 将CpG位点位置与丽蝇蛹集金小蜂（Nvit）基因组的外显子/内含子基因注释进行关联
5. 仅保留与基因特征存在重叠的CpG位点
6. 利用丽蝇蛹集金小蜂GO注释文件，为已映射的gene_id分配GO术语
7. 输出最终的基因-GO注释文件，作为背景集使用

输入文件
- erindata_v2.txt：Erin整理的甲基化矩阵文件
- un.dmls.timepoint.txt：仅包含时间点相关的差异甲基化CpG位点列表
- GCF_009193385.2_Nvit_psr_1.1_genomic_numbered_exons.txt：丽蝇蛹集金小蜂外显子/内含子注释文件
- Nasonia_PSR1.1_Gene_GO.txt：丽蝇蛹集金小蜂基因的GO注释文件

输出文件
- diff_methylatedcpgs_with_LOC.csv：已映射至注释基因特征的CpG位点文件
- diff_backgroundGOannotations.csv：用于富集分析的最终基因-GO背景集文件

软件需求
- 所需R包：data.table、dplyr、tidyr、readr、sqldf、GOstats、GSEABase、treemap
- 输入的基因组注释文件需包含带编号的外显子/内含子坐标及gene_id映射关系

备注
- 所有GO术语的证据代码均设置为"IEA"
- 未与基因特征重叠的CpG位点将被排除在背景集之外
- 该背景集后续将用于GO富集分析脚本（详见补充表S4分析）

联系方式
Eamonn Mallon，邮箱：[e-mail]